This study shows the effects of dietary supplementation with Lactobacillus acidophilus on the gut microbiota of broiler chickens challenged with Clostridium perfringens infection during a 21-day period according to pyrosequencing of the 16S ribosomal RNA gene. In a 2 × 2 factorial arrangement of treatments, 308 1-day-old male Arbor Acres broiler chicks were analyzed for the effects of the probiotic (groups without or with L. acidophilus supplementation), pathogen challenge (groups without or with C. perfringens), and the effects of interaction. The infection decreased the number of Observed species, Chao1, and ACE of ileal microbiota and increased Chao1 of cecal microbiota of broilers, whereas L. acidophilus supplementation decreased the Shannon index of the ileal microbiota. Shannon index and Simpson indices were lower in the ileal microbiota than in the cecal microbiota. In the ileal microbiota, the control group had higher relative abundance of Lachnospiraceae and Ruminococcaceae in comparison with the other groups; however, the relative abundance of Gammaproteobacteria was significantly higher in the challenge group than in the other groups. C. perfringens infection tended to increase lactate concentration and decreasedconcentrations of formate, acetate and propionate in the ileum; decreased isobutyrate concentration; and tended to decrease isovalerate concentration in the cecum. Besides, L. acidophilus supplementation increased the concentration of lactate and butyrate and decreased concentrations of formate and propionate in the ileum, and increased concentrations of lactate and valerate in the cecum. In conclusion, C. perfringens infection and/or dietary supplementation with L. acidophilus modulated the relative abundance of some bacteria taxa, and the L. acidophilus supplementation helped to restore the microbial community disrupted by C. perfringens infection.